Fluorescence microscopy of oligonucleotide probe arrays

摘要

We have been exploiting high density oligonucleotide arrays to carry out sequence analysis of genetic material from diverse sources. The method utilizes the hybridization of fluorophore labelled nucleic acids to the array and interpretation of the resulting spatial pattern of fluorescence. Our ability to obtain sequence information from the array is governed by the interplay of the synthesis and hybridization chemistry, the photophysics of the fluorophores and background interferences, and the performance of the fluorescence imaging system. The high photolithographic resolution and large usable area of the synthesis process and the presence of submonolayer coverages of fluorophores dictate that the fluorescence detection system meet several potentially conflicting performance criteria. High spatial resolution, high sensitivity, large field of view, low chromaticity and image distortion, and high dynamic range are required simultaneously. Suitable nucleic acid-fluorophore conjugates should have high absorption cross sections and emission quantum yields, low photobleaching quantum yields, and resistance to transient saturation under intense illumination. Our approaches to the design and photophysical characterization of the detection process will be discussed within the context of improving the volume of sequence information and detection limits.